Investigating metabolite-RNase communication.

Lead Research Organisation: University of Portsmouth
Department Name: Inst of Biomedical and Biomolecular Sc

Abstract

Within living cells a whole series of chemical reactions occur in order to provide the energy the cell needs to sustain life. This series of reactions is collectively known as a cell's metabolism. Understanding how metabolism is controlled within a cell is fundamentally important and is directly applicable to medical, environmental and biotechnological advances. At the present time, some aspects of how metabolism is controlled are understood, but we have recently discovered a whole new control mechanism of key importance.

It is already known that messenger molecules (RNA) within a cell play a role in controlling metabolism and that in turn, destroyer molecules (RNA degraders) in the cell keep the number of RNA molecules in check. Our studies have identified that one of the chemicals involved in metabolism, known as a metabolite, interacts with an RNA degrader and affects its ability to destroy RNA. Our work therefore indicates that a full feedback system exists within a cell, with metabolites altering the ability of RNA degraders to destroy RNA, which in turn affects cellular metabolism, which impacts metabolites, which then interact with RNA degraders and so the loop continues.

The aim of the proposed work is to investigate the newly identified interactions between metabolism and RNA-degraders in detail. Specifically, our objectives are to answer a number of key questions. What changes occur to the population of messenger molecules within the cell when this mechanism takes place and are some messenger molecules targeted earlier than others? By monitoring the population of messenger molecules can it be seen whether the mechanism changes once the RNA-degraders form larger complex structures with other RNA degraders? If we specifically change the metabolite-recognition site on the RNA-degrader, what happens to the population of messenger molecules and what can this tell us about the mechanism? Is this mechanism of communication between metabolism and RNA-degraders found in all types of cells from simple bacteria to complex animal cells? To answer these questions our research will use a comprehensive state-of-the-art toolset of proven practical and computational biological research techniques.

Understanding these additional details about the communication between metabolism and RNA-degraders allows us to take the next step towards realising the full impact of our recent discovery. In the longer term, such knowledge could allow scientists to artificially control metabolism within living cells. For example, simple bacterial cells play an important role in many industrial applications and this artificial metabolic control could optimise their use. This may potentially increase efficiency by reducing energy costs, increasing yields and reducing starting material requirements, all of economic and environmental value. Examples include exploitation within the pharmaceutical industry (e.g. more efficient drug production), the food industry (e.g. improvements in food production) and particularly in relation to environmental concerns (e.g. aiding biofuel production and bioremediation projects). In a similar manner, the artificial control of metabolism within animal cells has the potential to offer far reaching therapeutic benefits.

Technical Summary

The aim of this project is to expand the understanding of our recently discovered communicative link between central metabolism and ribonucleases (RNases) by investigating the details and level of conservation of the mechanism. Such RNases impact the abundance of many mRNA transcripts and structured RNAs, thereby affecting post-transcriptional gene expression. The control of RNase activities contributes to homeostasis and the response to environmental change and is thus likely to be affected by general metabolic activities. Our recent published data confirms that a key central metabolism intermediate in the Krebs cycle, citrate, affects the activity of the E. coli exoribonuclease, polynucleotide phosphorylase (PNPase) and that conversely, cellular metabolism is distributively affected by the activities of PNPase and the multi-enzyme RNA degradosome complex.

With our recent fundamental discovery as a basis, this proposal seeks to investigate the newly identified regulatory interactions in more detail. We will investigate the impact of metabolite-PNPase communication at the transcript level using the standard transcriptomic techniques of microarray analysis and real time RT-PCR. Specifically, we will validate and quantify our earlier microarray analysis and explore the rates of transcript decay. We will then determine whether metabolite-PNPase communication is altered when PNPase is in complex form (i.e. as part of the RNA degradosome) or following mutation of a PNPase-metabolite-binding site. Finally, we will identify whether the metabolite-PNPase communicative link represents a conserved mechanism through the identification and characterisation of homologous RNase-metabolite relationships using computational, biochemical, biophysical and structural techniques. The implications of this work stand to be significant, aiding our appreciation of this recently discovered mechanism and providing us with a comprehensive understanding of metabolite-RNase communication.

Planned Impact

The potential impact of the proposed work broadly falls into the three main areas:

1) Impacting the field of systems biology
With significant transcriptomic data generated during this proposal, a key immediate impact of research will be within the systems biology field. Our work detailing the specific transcripts altered by the metabolite-RNase communicative link will form the basis for the development of metabolite-RNase-transcript models. It will thus be valuable to computational modelling specialists in universities and research institutes, who will be able to use our transcriptomic data to create models for in silico testing of cellular scenarios, which could subsequently be tested experimentally, potentially by metabolomics specialists. Refinement of such models will ultimately allow it to be possible to predict the effect on the transcriptome of a metabolic change within a cell and hence conduct targeted manipulations of the metabolome with therapeutic and industrial benefits in mind. Overall, this will sustain the knowledge economy by supporting the UK's leading role in Systems biology.

2) Instigation of new research programmes
The results of the proposed work will impact research groups in the wider field of post-transcriptional gene regulation control, specifically within academic disciplines such as transcriptomics, metabolomics, biochemistry, biophysics, structural biology, molecular biology, microbiology and cell biology. Such researchers can be found within universities, research institutes, charities and industry. Our proposed work will support the instigation of new research programmes that build on our findings; allowing a further characterisation of the mechanism and a full understanding of its cellular impact to be undertaken. Specifically, should our research identify that the metabolite-RNase mechanism that we have discovered in the bacteria, E. coli, is conserved in the other domains of life i.e. archaea and eukaryotes, then a key investigational element will be to undertake in vivo studies in such organisms. This research will provide new information and scientific advancement thereby enhancing the UK's knowledge economy. Maintaining and expanding research in this manner will contribute towards the skills of researchers involved in such studies. Instigating new research programmes will therefore not only benefit scientific research in knowledge terms but will also provide the UK workforce with trained, highly skilled, researchers.

3) Exploitation within the metabolic engineering domain
A major longer term future potential application of this work lies within the metabolic engineering domain. This research to clarify the metabolite-RNase communicative link could provide the basis for manipulation of metabolic processes in bacteria to artificially control RNA decay rates. This metabolic intervention could subsequently be used to optimise cellular processes; increasing efficiency thereby reducing energy costs, increasing yields and reducing starting material requirements - all of which have the potential to offer economic and environmental benefits. This exploitation route would therefore be expected to be highly attractive to the private sector, specifically:
- to the pharmaceutical industry, for more efficient synthetic drug production such as insulin
- to the food industry, for improved food production
- to address environmental challenges, such as improved biofuel production and bioremediation
The exploitation of scientific knowledge could support the creation of new processes and products, contributing to the economic prosperity for the companies involved and the economic competitiveness of the UK.
 
Title 3D Printed Molecular Structures 
Description Working with Dr Darren Gowers at the University of Portsmouth, who runs the 3D molecular models printing service, the molecular structures of a number of molecules of interest to our research were generated. By incorporating tiny magnets into the structures it was possible to generate molecular models of protomers (single units) that could be assembled into their native oligomeric states; thus yielding flexible molecular models that could be disassembled and reassembled to appreciate the interactions involved in creating multi-biomolecule complexes. 
Type Of Art Artwork 
Year Produced 2014 
Impact These models provide hands-on aids when presenting our research findings to both scientific and general public audiences. This has allowed potentially complex molecular details to be communicated clearly and easily and has supported my research team in maximising the accessibility of our research to the wider scientific and lay communities. 
 
Description Background and Significant New Knowledge Generated:
Within living cells a whole series of chemical reactions occur in order to provide the energy the cell needs to sustain life. This series of reactions is collectively known as a cell's metabolism. Understanding how metabolism is controlled within a cell is fundamentally important and is directly applicable to medical, environmental and biotechnological advances. It is already known that messenger molecules (RNA) within a cell play a role in controlling metabolism and that in turn, destroyer molecules (RNA degraders known as ribonucleases) in the cell keep the number of RNA molecules in check.

Underpinning the work in this grant was our recent discovery of a whole new control mechanism of key importance in bacteria. Specifically, using E. coli as a model bacterial organism, we identified that one of the chemicals involved in metabolism, known as a metabolite called citrate, interacts with a ribonuclease, called PNPase, and affects its ability to destroy RNA. Research funded by this grant has exposed the broad implications of this initial finding; providing evidence for the existence of citrate-mediated inhibition of PNPase not only across the bacterial kingdom, but of PNPase homologs in all three domains of life, i.e. in archea and eukaryotes. Our important discovery therefore highlights that the communicative link between ribonuclease activity and central metabolism may have been conserved through the course of evolution.

Further investigating the implications of our discovery, of the wide range of metabolites tested, we identified that only a handful (e.g. TCA Cycle compounds: acetyl-coA, cis-aconitate and succinyl-coA; second messenger molecule: ppGpp; nucleotides: GTP) had the ability to impact PNPase activity. Computational studies to explore the predicted metabolite-ribonuclease binding modes identified common features in the metabolites responsible for inhibiting ribonuclease activity through active site occlusion. Experimental studies verified computational predictions and in order to delve into the molecular detail of this metabolite-communicative link, a novel assay was developed to allow the assessment of PNPase activity in real time.

At present, publication of our findings on the conservation of the metabolite-communicative link across all three domains of life has been completed. Publications on broader involvement of TCA cycle metabolites, second messenger molecules and nucleotides in PNPase regulation are being prepared. A separate manuscript documenting the novel assay developed during this study is being prepared.

Important New Research Questions Opened Up:
This study is only the tip of the iceberg in terms of understanding metabolite-ribonuclease communication in cells. Our key discovery that the citrate-PNPase communicative link is conserved across all three domains of life sets the foundation upon which to explore this more broadly. Are other ribonucleases regulated by metabolites? How widespread are metabolite-ribonuclease communicative links conserved across the three domains of life? Can the links between cellular metabolic changes and ribonuclease impact on the RNA population be unravelled? Can such knowledge provide the basis for manipulation of metabolic processes in cells which could be exploited for metabolic intervention purposes, such as to optimising cellular processes, increasing efficiency to energy costs, increasing yields and reducing starting material requirements? Does knowledge of metabolite-ribonuclease communication have implications for disease treatment?

New or improved research methods or skills developed:
The researchers involved in the study have all developed a range of highly employable scientific skills spanning experimental and computational methods. In particular, our research has demonstrated the value of using in silico molecular docking to accurately guide experimental testing. As well as expanding and honing such scientific skills, transferable skills development has also been a key element of the project. Attendance at both scientific and broader transferable skills training courses has supported the personal development of the researchers working on the project and their attendance at conferences has raised not only their personal profiles as researchers but also the profile of our discoveries.

The novel experimental assay developed as part of this study has the potential for broad utility as a screening tool for identification of PNPase inhibitor compounds; or indeed determining phosphorolytic degradation of poly(A) tails by any 3'-5' exoribonuclease. The identification of such compounds, for use as chemical tools, would provide a means for in vivo testing of activity of such RNases within a cellular context. They could also be used in artificially controlling ribonuclease activity, for example for bacterial metabolic engineering purposes.

Particularly noteworthy new research networks/collaborations/partnerships, or combinations of these;
We are presently working with a company (Bellbrook Labs) to publish the novel high throughput approach we have developed for determining phosphorolytic degradation of poly(A) tails by PNPase (or any 3'-5' exoribonuclease) which has the potential for use as a broad screening tool for identification of inhibitor compounds. This will support wide accessibility of the research community to the novel assay as well as ensure provision of the reagents for ease of assay testing, though provision of the re-purposed commercial assay kit via Bellbrook Labs.

To expand this research, early discussions with eukaryotic experts are underway; supporting discussions into the next steps to take to explore RNase-metabolite communication within the context of model organisms such as Xenopus and Drosophila.

Summary
Overall, the research conducted as part of this grant have highlighted a communicative link to be present between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.
Exploitation Route - Utilisation of novel assay: The novel assay developed as part of this work for determining phosphorolytic degradation of poly(A) tails by PNPase (or any 3'-5' exoribonuclease) has the potential for use as a broad screening tool for identification of inhibitor compounds. Such compounds would be of value as tool molecules for use in unravelling/characterising RNase activities within a cellular context, for possibly identifying potential therapeutic lead molecules which target such RNases for medicinal purposes, and/or for development of molecules for potential control of activity within a metabolic engineering setting.
- Instigation of new research programmes: The discoveries made as part of this work will support the instigation of new research programmes that build on and expand our findings; allowing a further characterisation of the metabolite-RNase mechanism and a full understanding of its cellular impact to be undertaken. This research will provide new information and scientific advancement thereby enhancing the UK's knowledge economy. Maintaining and expanding research in this manner will contribute towards the skills of researchers involved in such studies. Instigating new research programmes will therefore not only benefit scientific research in knowledge terms but will also provide the UK workforce with trained, highly skilled, researchers.
- Exploitation within the metabolic engineering domain: The outputs of this work, characterising and highlighting the importance of metabolite-RNase communication, could provide the basis for manipulation of metabolic processes in bacteria to artificially control RNA decay rates. Such metabolic intervention could subsequently be used to optimise cellular processes; increasing efficiency thereby reducing energy costs, increasing yields and reducing starting material requirements - all of which have the potential to offer economic and environmental benefits.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings of the grant have resulted in a number of publications (some already published and some still in the process of being published). Additionally, a novel high throughput approach for determining phosphorolytic degradation of poly(A) tails by a 3'-5' exoribonuclease has been developed, in conjunction with Bellbrook Labs, through modification and re-purposing of one of their commercial assay kits. Being useful for researchers in the field, this has the potential to broaden kit utility, with associated economic impact to the company.
 
Description University of Portsmouth IBBS PhD Studentship
Amount £55,000 (GBP)
Organisation University of Portsmouth 
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 09/2016
 
Title High throughput novel approach for determining phosphorolytic degradation of poly(A) tails by a 3'-5' exoribonuclease 
Description As part of this research a novel method has been developed which allows the high throughput testing of the phosphorolytic degradation of poly(A) tails by a 3'-5' exoribonuclease. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact A manuscript is being prepared so that the method can be used more widely by those in the field. 
 
Description Collaboration with Bellbrook Labs 
Organisation Bell Brook Labs LLC
Country United States 
Sector Private 
PI Contribution Myself and my research team provided expertise on the topic of the research project.
Collaborator Contribution Bellbook Labs provided support and advice with respect to our work to development and validate a novel high throughput approach applicable to the research project which involved modifying and re-purposing one of their assay kits.
Impact A manuscript is currently being prepared to document the novel high throughput approach, applicable to the research project, that was developed as part of this collaboration.
Start Year 2015
 
Description Collaboration with Dr Kenny McDowall at Leeds University 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration involved researchers in my team conducting in vitro tests of potential inhibitor molecules identified and provided by Dr McDowall's group following computational analysis.
Collaborator Contribution Potential inhibitor molecules were identified and provided by Dr McDowall's group for interaction testing by researchers within my team.
Impact A paper of the findings resulted from this collaboration. My team provided in vitro molecular interaction and activity testing expertise whilst Dr McDowall's team also provided expertise in activity testing as well as computational analysis.
Start Year 2009
 
Description Collaboration with Dr Paul Cox at the University of Portsmouth (2013 - Still Active) 
Organisation University of Portsmouth
Department School of Health Sciences and Social Work
Country United Kingdom 
Sector Academic/University 
PI Contribution Myself and my research team provide expertise in the topic area of investigating metabolite-RNase communication.
Collaborator Contribution My collaborator provides expertise in molecular docking methods which were undertaken as part of this research project.
Impact A manuscript documenting our collaborative research results has recently been accepted by Nucleic Acids Research.
Start Year 2013
 
Description Collaboration with Prof. Matt Guille, European Xenopus Resource Centre, University of Portsmouth 
Organisation University of Portsmouth
Department European Xenopus Resource Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Myself and my team provide expertise in the subject area of the research project.
Collaborator Contribution Prof. Guille provides expertise in a eukaryotic model organisms. Early preliminary work to explore conservation of the metabolite-RNase communicative link, identified in prokaryotes and Archea, within eukaryotes is underway.
Impact Undergraduate project students have been co-supervised by Profs Callaghan and Guille, as well as a current PhD student. All students have been involved in delivering data and compiling reports on the collaborative topic that stems from this research grant.
Start Year 2013
 
Description Invited seminar on the Molecular Control of RNA Metabolism - Kent 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker at the University of Kent, April 2014.

Effective networking, building collaborative relationships.
Year(s) Of Engagement Activity 2014
 
Description Invited seminar on the Molecular Control of RNA Metabolism - Southampton 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker, University of Southampton, April 2014.

Effective networking, building collaborative relationships.
Year(s) Of Engagement Activity 2014
 
Description Invited seminar presentation on the Molecular Control of RNA Metabolism - Bath 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Invited seminar speaker at Bath University, April 2011.

Effective networking, building collaborative relationships.
Year(s) Of Engagement Activity 2011
 
Description Maintaining an Active Online Presence 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My group has an active Twitter account with around 200 followers. We publish highlights from our research, outreach and engagement activities.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Promoting PG study 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Presentation by members of my research team at various departmental events for undergraduates to promote engagement in postgraduate study. this involved the individuals highlighting their research work, including their day to day work, opportunities for collaboration and engagement as well as their outputs and impact.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Science Fairs 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Myself and my team have participated in supporting a number of Science Fairs in the region, engaging with attendees to promote science and the research we undertake.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Myself and my team regularly support University Open days. Activities can be many and varied, including giving talks, presenting posters, running hands-on laboratory demonstrations and engaging in question and answer sessions. There are usually a number of these events per year, with over 100 participants (schools and college students, sometimes accompanied by a parent/guardian) attending each event. Feedback from such events has highlighted our success in inspiring the next generation of scientists and has been specifically linked to an increase in the number of students applying to study Biochemistry over the last few years.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013
 
Description University of Portsmouth Graduate School Induction 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Presentation to postgraduate students as part of their Graduate School Induction regarding on-going research. The presenter was awarded a prize for the best presentation, acknowledging her ability to communicate her research to a broad audience; including those both within her research area as well as those from a range of non-scientific disciplines across the institution.
Year(s) Of Engagement Activity 2016